KR102511149B1 - Bee venom sterilization process using E-beam and verification of sterilization process using Hepatitis A Virus - Google Patents

Abstract

The present invention relates to a method of irradiating bee venom with an E-beam to sterilize the virus in the bee venom, and verifying whether the virus is sterilized by the sterilization process using hepatitis A virus. Microbes present in bee venom can be sterilized by using the method of sterilizing viruses in bee venom, which includes the step of irradiating bee venom with an E-beam of the present invention, and through this, unlike conventional sterilization processes, bee venom It does not change the active ingredient of the bee venom that is safe for the human body and can be sterilized. In addition, by using the process of confirming sterilization using the hepatitis A virus of the present invention, it is possible to effectively confirm that all viruses in the bee venom are sterilized, thereby manufacturing a safe bee venom whose sterilization has been verified.

Description

Bee venom sterilization process using E-beam and verification of sterilization process using Hepatitis A Virus}

The present invention relates to a method of irradiating bee venom with an E-beam to sterilize the virus in the bee venom, and verifying whether the virus is sterilized by the sterilization process using hepatitis A virus.

Bee venom, one of the animal sources produced by bees, can contain unwanted and potentially dangerous contaminants such as viruses, bacteria, yeasts, molds, mycoplasma and parasites. Since bee venom can be used for administration to the human body, it is very important to remove contaminants that may exist in bee venom.

Methods conventionally known as sterilization processes for animal raw materials include heating, filtration, and adding chemical inactivators or sensitizers to the product. Heat treatment, which heats the product at approximately 60° C. for about 70 hours, can destroy up to about 50% of the activity of contaminating microorganisms, but may cause damage to sensitive products. The filtration process physically filters the product to remove contaminants. However, this method can remove the active ingredient of bee venom having a high molecular weight, and in some cases, small viruses may remain by filtration. The chemical sterilization process involves UV or ionization to produce free radicals that bind to the DNA/RNA of the virus and break the chemical bonds to the DNA/RNA backbone of the virus in such a way that the virus can no longer replicate. It is the process of adding harmful reagents that are activated by radiation. It has been pointed out that this process may cause toxicity, side effects may occur when administered to humans, and problems may cause modification of the active ingredient of bee venom. Therefore, there is a need for a virus sterilization process in bee venom that does not change the active ingredient of bee venom and can ensure the safety of bee venom that can be used as a medicine.

On the other hand, as a conventional test method for confirming the presence of a contaminant in bee venom, a method of individually detecting the contaminating microorganisms for bacteria, viruses, and fungi suspected of being contaminated and confirming whether or not they are contaminated by individual microorganisms has been disclosed. . However, it has been pointed out that such a process cannot confirm contamination by an unexpected contaminant, and a technique capable of confirming the absence of a virus in bee venom that solves this problem has been required.

Accordingly, the present inventors studied a safe sterilization process while effectively sterilizing the virus in the bee venom without causing a change in active ingredients, and studied a method for effectively confirming that sterilization was sterilized by the sterilization process, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for sterilizing viruses in bee venom, which includes irradiating E-beam to bee venom, and to determine whether sterilization is performed using the step of irradiating E-beam and hepatitis A virus. It is to provide a method for preparing bee venom, including the step of identifying, and sterilized bee venom thereby.

In order to achieve the above object, the present invention provides a method of sterilizing hepatitis A virus in bee venom, comprising irradiating the bee venom with an electron beam.

In addition, the present invention includes (a) irradiating E-beam to bee venom; and (b) detecting the hepatitis A virus in the bee venom.

Microbes present in bee venom can be sterilized by using the method of sterilizing viruses in bee venom, which includes the step of irradiating bee venom with an E-beam of the present invention, and through this, unlike conventional sterilization processes, bee venom It does not change the active ingredient of the bee venom that is safe for the human body and can be sterilized. In addition, by using the process of confirming sterilization using the hepatitis A virus of the present invention, it is possible to effectively confirm that all viruses in the bee venom are sterilized, thereby manufacturing a safe bee venom whose sterilization has been verified.

1 is a schematic view showing a manufacturing process of bee venom.
2 is a diagram confirming that all hepatitis A viruses are killed by irradiating E-beam to a sample spiked with hepatitis A virus in bee venom.
3 is a diagram confirming that all porcine parvovirus is killed by irradiating E-beam to a sample spiked with porcine parvovirus in bee venom.

The present invention provides a method of sterilizing hepatitis A virus in bee venom, comprising irradiating the bee venom with an E-beam.

Hereinafter, the present invention will be described in more detail.

In the present invention, 'bee venom' is venom from the ovipositor of bees.

The bee venom of the present invention may be live bee venom, dried bee venom, bee venom extract or purified bee venom. Live bee venom is undried or purified, has specific gravity of 1.1 to 1.3, acidity (pH) in the range of 5.2 to 5.5, and 70 to 80% by weight may be composed of proteins or peptides. The bee venom extract can be extracted using various extraction methods known in the art, and is preferably water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), such lower alcohol and water. It may be obtained by using a mixed solvent, acetone, ethyl acetate, or butylene glycol as an extraction solvent. The purified bee venom may be obtained through a conventional purification process in addition to the extract using the above extraction solvent, or may be purified bee venom by separation using a filtration membrane, separation by various chromatographic methods, or purification methods.

The bee venom of the present invention can be administered to a subject to improve the body's growth rate, immune function, and pain treatment function.

In the present invention, 'sterilization' may be reduction of biological contaminants or microorganisms, preferably retroviruses, herpes viruses, paramyxoviruses, cytomegaloviruses, A such as hepatitis virus, hepatitis B virus, hepatitis C virus, pox viruses, toga viruses, Epstein-Barr virus and parvoviruses virus; bacteria such as Escherichia, Bacillus, Campylobacter, Streptococcus and Staphalococcus; parasites such as Trypanosoma; malaria parasites including Plasmodium species; leaven; mold; mycoplasma; And it may be reduction of any one or more contaminants selected from the group consisting of prions, more preferably reduction of any one or more viruses selected from the group consisting of bovine herpes virus, hepatitis A virus and porcine parvovirus. .

In the present invention, the 'sterilization method' may be any method using effective radiation for sterilization of bee venom. Preferably the radiation may be small particles including E-beam radiation.

The 'E-beam (electron beam)' of the present invention may be an E-beam irradiated with a dose of 20 to 100 kGy, preferably an E-beam irradiated with a dose of 25 to 50 kGy. When the E-beam is irradiated with an irradiation amount of 20 kGy or less, the virus in the bee venom may not be sterilized, and when the E-beam is irradiated with an irradiation amount of 100 kGy or more, there is a risk of deterioration of bee venom color due to high power.

The 'E-beam' of the present invention does not cause a change in active ingredients in bee venom, and preferably contains melittin, apamin, mast cell degranulation peptides (MCD-Peptide, Mast Cell Degranulation Peptide, Op), protease inhibitor, Secarpin, Tertiapin, or Procamine, and more preferably does not cause melittin change.

According to the 'method for sterilizing hepatitis A virus in bee venom' of the present invention, microorganisms present in bee venom can be sterilized without changing the active ingredient of bee venom.

In addition, the present invention includes (a) irradiating E-beam to bee venom; and (b) detecting the hepatitis A virus in the bee venom.

The E-beam in step (a) of the present invention may be an E-beam irradiated with an irradiation amount of 20 to 100 kGy, preferably an E-beam irradiated with an irradiation amount of 25 to 50 kGy. When the E-beam is irradiated with an irradiation amount of 20 kGy or less, the virus in the bee venom may not be sterilized, and when the E-beam is irradiated with an irradiation amount of 100 kGy or more, there is a risk of deterioration of bee venom color due to high power.

In the present invention, the detection in step (b) is performed by polymerase chain reaction (PCR), ligase chain reaction (LCR), Gap-LCR (Gap-ligase chain reaction), repair chain reaction, transcription -mediated amplification (TMA), self sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence priming polymerase chain reaction (CP-PCR), arbitrary priming polymerase chain reaction (AP-PCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, loop-mediated thermostatic amplification (LAMP), multiplex PCR, double Polymerase chain reaction (nested-PCR), single tube nested-PCR, reverse transcription-polymerase chain reaction (RT-PCR), inverse polymerase chain reaction (inverse PCR), real-time polymerase At least one method selected from the group consisting of chain reaction (RT-PCR), real-time polymerase chain reaction quantitative test (RQ-PCR), capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement and phosphorescence measurement It may be performed using, preferably, a reverse transcription-polymerase chain reaction (RT-PCR) method and gel electrophoresis, but is not limited thereto.

In the present invention, the method for preparing the sterilized bee venom may further include, prior to step (a), spiking the hepatitis A virus of step (b) into bee venom. If the step of spiking the hepatitis A virus in step (b) is further included, the hepatitis A virus spiked in step (b) is detected and if the hepatitis A virus is not detected, the ( It can be confirmed that the virus in the bee venom is sterilized by step a).

In the present invention, the model virus is a virus capable of ensuring sterilization of an unknown or unidentified virus, and may be a virus itself or a virus most similar to it that may contaminate bee venom, preferably bovine herpes virus, It may be any one or more viruses selected from the group consisting of hepatitis A virus and porcine parvovirus.

According to the 'method for producing sterilized bee venom' of the present invention, bee venom verified to be sterilized of viruses in bee venom can be prepared without changing the active ingredient in bee venom.

In addition, the present invention provides a sterilized bee venom prepared by the method for preparing sterilized bee venom.

The sterilized bee venom of the present invention is characterized in that the virus in the bee venom is verified to be sterilized, and the active ingredient of the bee venom remains unchanged even after the sterilization process.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples.

Example 1. Selection and cultivation of model virus to verify virus sterilization effect

1.1 Selection of model virus to verify virus sterilization effect

The following criteria were considered to select model viruses for verifying the virus sterilization effect of the virus sterilization process in bee venom.

(i) The virus to be used for verification of virus contamination in bee venom must be the virus itself or the most similar virus that can contaminate bee venom. In addition, by selecting viruses of various physical and chemical properties, it should be possible to show that the virus sterilization process to be verified can generally sterilize all types of viruses.

(ii) The selected virus should preferably be highly resistant to physicochemical treatment to ensure sterilization of other unknown or unidentified viruses.

(iii) the virus should be as readily assayable as possible and capable of being cultured at high concentrations;

(iv) The selected virus should present a low risk of infection to the experimenter during the testing period being verified.

Considering these points, bovine herpes virus (BHV), hepatitis A virus (HAV), and porcine parvovirus (PPV) are the most model viruses to verify viruses in the bee venom. As it was suitable, it was selected as a model virus to check whether the virus was sterilized in the bee venom.

1.2 Cultivation of bovine herpes virus

MDBK NBL-1 (ATCC CCL-22) cells were placed in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in a 37°C, 5% CO ₂ incubator. After infecting bovine herpes virus (ATCC VR-188) to monolayer MDBK NBL-1 (ATCC CCL-22) cells cultured in a T-150 flask, virus culture medium (DMEM medium containing 2% fetal bovine serum) 35 ℃, 5% CO ₂ Put the cytopathic effect (cytopathic effect, CPE) was observed periodically while culturing in an incubator. When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were disrupted by freezing and thawing three times, and the cell debris was removed by centrifugation. The centrifugal supernatant was filtered with a 0.45 μm filter and then subdivided and stored at -70 ° C.

1.3 Culture of hepatitis A virus

FRhK-4 (ATCC CRL-1688) cells were placed in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in a 37°C, 5% CO ₂ incubator. After infecting monolayer FRhK-4 (ATCC CRL-1688) cells cultured in a T-150 flask with hepatitis A virus (ATCC VR-1402), virus culture medium (DMEM medium containing 2% fetal bovine serum) 35 ℃, 5% CO ₂ Put the cytopathic effect (cytopathic effect, CPE) was observed periodically while culturing in an incubator. When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were disrupted by freezing and thawing three times, and the cell debris was removed by centrifugation. After filtering the centrifugal supernatant with a 0.45 μm filter, it was subdivided and stored at -70 ° C.

1.4 Cultivation of porcine parvovirus

MPK (ATCC CCL-166) cells were placed in a cell culture medium (DMEM medium containing 10% fetal calf serum and L-glutamine) and cultured in a 37°C, 5% CO ₂ incubator. After infecting single-layer MPK (ATCC CCL-166) cells cultured in a T-150 flask with porcine parvovirus (ATCC VR-742), add virus culture medium (DMEM medium containing 2% fetal bovine serum) 35 ℃, 5% CO ₂ While culturing in an incubator, the cytopathic effect (cytopathic effect, CPE) was observed periodically. When the cytopathic effect was clearly observed, the culture medium and cell debris were collected, the cells were disrupted by freezing and thawing three times, and the cell debris was removed by centrifugation. After filtering the centrifugal supernatant with a 0.45 μm filter, it was subdivided and stored at -70 ° C.

Example 2. Determination of E-beam dose for virus inactivation process in bee venom

2.1 Confirmation of sterilization effect according to E-beam irradiation amount for samples spiked with bovine herpes virus in bee venom

Among the bee venom manufacturing processes, the E-beam irradiation process is a sterilization process in which lyophilized bee venom powder is irradiated with 25 kGy E-beam, and is the final process to inactivate infectious risk factors. For the E-beam, thermal's E-beam was prepared, and for the bee venom, domestically available purified bee venom was purchased and used. A scaled-down process of the E-beam irradiation process was designed to verify the virus inactivation effect in the E-beam irradiation process. Experiments were conducted to confirm the optimal E-beam irradiation amount capable of exhibiting virus inactivation in the bee venom scale-down process. As a control, a purified bee venom sample without any treatment and bovine herpes virus cultured in Example 1.2 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with bovine herpes virus, a model virus cultured in Example 1.2, at a concentration of 1 x 10 ⁷ cfu/ml was prepared, and the purified bee venom spiked with the virus was prepared. Samples that were freeze-dried and sterilized using an E-beam at an irradiation dose of 1 kGy, 5 kGy, 10 kGy, and 25 kGy were prepared, respectively. The presence of bovine herpesvirus in the sample was confirmed by performing RT-PCR using the Bovine herpesvirus 1 putative fibronectin binding protein genesig® Advanced Kit (Primerdesign TM Ltd). The reduction ratio in the designed scale-down process is shown in Table 1, and the results of bovine herpes virus detection in each experimental group and control group are shown in Table 2.

lane processing conditions Detection of bovine herpes virus One bovine herpes virus sample detection 2 Freeze-dried sample of purified bee venom spiked with bovine herpes virus that has not undergone the E-beam sterilization process detection 3 Purified bee venom freeze-dried sample spiked with bovine herpes virus that has undergone the 1 kGy E-beam sterilization process detection 4 Purified bee venom freeze-dried sample spiked with bovine herpes virus that has undergone a 5 kGy E-beam sterilization process detection 5 Purified bee venom freeze-dried sample spiked with bovine herpes virus subjected to 10 kGy E-beam sterilization process detection 6 Purified bee venom freeze-dried sample spiked with bovine herpes virus that has undergone the 25 kGy E-beam sterilization process non-detection 7 Unprocessed purified bee venom freeze-dried sample non-detection

2.2 Confirmation of sterilization effect according to E-beam irradiation amount for samples spiked with hepatitis A virus in bee venom

In order to confirm the optimal E-beam irradiation amount capable of exhibiting virus inactivation in the scale-down process, an experiment was additionally performed targeting hepatitis A virus. As a control, a purified bee venom sample without any treatment and the hepatitis A virus cultured in Example 1.3 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with hepatitis A virus, a model virus cultured in Example 1.3, at a concentration of 1 x 10 ⁷ cfu/ml was prepared, and purified bee venom spiked with the virus Freeze-dried and sterilized samples were prepared using E-beams of 1 kGy, 5 kGy, 10 kGy, and 25 kGy, respectively. The following experiments were performed to detect hepatitis A virus in samples.

For RNA isolation, 1 g of untreated purified bee venom sample, 0.5 mg of purified bee venom freeze-dried sample spiked with hepatitis A virus that had not undergone E-beam sterilization, and hepatitis A virus spiked with E-beam sterilized 0.5 mg of the purified bee venom freeze-dried sample was added to 5 mL, 1 mL, and 1 mL of TRIzol Reagent (Thermo Fisher Scientific, CA, USA), homogenized, and reacted at room temperature for more than 15 minutes. ) was mixed with 200 μL, reacted at room temperature for 5 minutes, and centrifuged (12000 g, 15 minutes, 4° C.). The supernatant was transferred to a new tube and reacted with 500 μL of isopropanol (Sigma-aldrich) at room temperature for 10 minutes to precipitate RNA, followed by centrifugation (12000 g, 10 minutes, 4°C), and after removing the supernatant, 80% ethanol It was washed using and centrifuged (12000 g, 5 minutes, 4 ℃), the precipitate (pellet) was dissolved in DEPC treated water (WelGENE, Korea) and used in the experiment at a concentration of about 1 μg / μL. After synthesizing 1 μg of the isolated RNA into cDNA using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Korea), 2 μL of cDNA and 10 pmoles of primer were added in a 20 μL reaction using AccuPower PCR Premix from Bioneer (Korea). RNA of hepatitis A virus was detected by performing RT-PCR (TP3300, Takara, Japan) as much as possible. The reaction conditions were denatured at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 1 minute, and amplification was performed at 72°C for 5 minutes. Amplification products were electrophoresed at 100V on a 1.2% agarose gel using 0.5 × TAE solution, and expression was confirmed using Safe-Pinky DNA Gel Staining solution (GenDEPOT). Primer sets used for RT-PCR are shown in Table 3, and detection results are shown in Figs. 2 and 4.

lane processing conditions Whether hepatitis A virus was detected One Hepatitis A virus sample detection 2 Freeze-dried sample of purified bee venom spiked with hepatitis A virus that has not undergone the E-beam sterilization process detection 3 Purified bee venom freeze-dried sample spiked with hepatitis A virus that has undergone a 1 kGy E-beam sterilization process detection 4 Purified bee venom freeze-dried sample spiked with hepatitis A virus that has undergone the 5 kGy E-beam sterilization process detection 5 A freeze-dried sample of purified bee venom spiked with hepatitis A virus that has undergone a 10 kGy E-beam sterilization process detection 6 A freeze-dried sample of purified bee venom spiked with hepatitis A virus that has undergone a 25 kGy E-beam sterilization process non-detection 7 Unprocessed purified bee venom freeze-dried sample non-detection

2.3 Confirmation of sterilization effect according to E-beam irradiation amount for samples spiked with porcine parvovirus in bee venom

In order to confirm the optimal E-beam irradiation amount capable of exhibiting virus inactivation in the scale-down process, an experiment was additionally conducted on porcine parvovirus. As a control, a purified bee venom sample without any treatment and porcine parvovirus cultured in Example 1.4 were prepared. As an experimental group, a freeze-dried sample of purified bee venom spiked with porcine parvovirus, a model virus cultured in Example 1.4, at a concentration of 1 x 10 ⁷ cfu/ml was prepared, and the purified bee venom spiked with the virus was prepared. Samples that were lyophilized and sterilized using E-beam at an irradiation dose of 1 kGy, 5 kGy, 10 kGy, and 25 kGy were prepared, respectively. The following experiments were performed to detect porcine parvovirus in samples.

For RNA isolation, 1 g of untreated purified bee venom sample, purified bee venom spiked with porcine parvovirus that had not undergone E-beam sterilization, 0.5 mg freeze-dried sample, purified bee venom spiked with porcine parvovirus that had not undergone E-beam sterilization 0.5 mg of freeze-dried sample was added to 5 mL, 1 mL, and 1 mL of TRIzol Reagent (Thermo Fisher Scientific, CA, USA), homogenized, and reacted at room temperature for more than 15 minutes. were mixed and reacted at room temperature for 5 minutes, followed by centrifugation (12000 g, 15 minutes, 4°C). The supernatant was transferred to a new tube and reacted with 500 μL of isopropanol (Sigma-aldrich) at room temperature for 10 minutes to precipitate RNA, followed by centrifugation (12000 g, 10 minutes, 4°C), and after removing the supernatant, 80% ethanol It was washed using and centrifuged (12000 g, 5 minutes, 4 ℃), and the precipitate (pellet) was dissolved in DEPC treated water (WelGENE, Korea) and used in the experiment at a concentration of about 1 μg / μL. After synthesizing 1 μg of the isolated RNA into cDNA using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Korea), 2 μL of cDNA and 10 pmoles of primer were added in a 20 μL reaction using AccuPower PCR Premix from Bioneer (Korea). RT-PCR (TP3300, Takara, Japan) was performed as much as possible. The reaction conditions were denatured at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 1 minute, and amplification was performed at 72°C for 5 minutes. The amplification product was electrophoresed at 100 V on a 1.2% agarose gel using a 0.5 × TAE solution, and detection of porcine parvovirus was confirmed using Safe-Pinky DNA Gel Staining solution (GenDEPOT). Primer sets used for RT-PCR are shown in Table 5, and detection results are shown in Figs. 3 and 6.

Lane processing conditions Porcine parvovirus detection One Porcine parvovirus sample detection 2 Purified bee venom freeze-dried sample spiked with porcine parvovirus that has not undergone the E-beam sterilization process detection 3 Purified bee venom freeze-dried sample spiked with porcine parvovirus that has undergone the 1 kGy E-beam sterilization process detection 4 Purified bee venom freeze-dried sample spiked with porcine parvovirus that has undergone the 5 kGy E-beam sterilization process detection 5 Purified bee venom freeze-dried sample spiked with porcine parvovirus that has undergone the 10 kGy E-beam sterilization process detection 6 Purified bee venom freeze-dried sample spiked with porcine parvovirus that has undergone the 25 kGy E-beam sterilization process non-detection 7 Unprocessed purified bee venom freeze-dried sample non-detection

As shown in Tables 2, 4, and 6, it was confirmed that no spiked bovine herpes virus, hepatitis A virus, or porcine parvovirus was detected in the purified bee venom subjected to the E-beam sterilization process at an irradiation dose of 25 kGy. did

Therefore, when the bee venom was subjected to the E-beam sterilization process, it was confirmed that all viruses capable of contaminating the bee venom, represented by bovine herpes virus, hepatitis A virus, and porcine parvovirus, were all sterilized.

Example 3. Changes in melittin content in bee venom according to virus inactivation process in bee venom

Unlike other virus inactivation processes, the sterilization process of the present invention does not cause denaturation of major active ingredients in bee venom, and an experiment was conducted to confirm this. Melittin was selected as the main active ingredient, and a high pressure sterilization method and an alcohol sterilization method were selected as a virus inactivation process for comparison.

3.1 Measurement of changes in melittin content in bee venom by high-pressure sterilization process

As a control, purified bee venom commercially available in Korea that had not undergone a sterilization process was prepared without treatment. As an experimental group, samples obtained by dissolving purified bee venom in 100% distilled water to make 1 mg/mL and then heating at 121°C for 5 minutes, 10 minutes, 15 minutes, and 20 minutes using a sterilizer were set as the experimental group, respectively. Melittin content analysis for each experimental group was measured using a UPLC I class model equipped with a diode array detector from Waters (Minneapolis, MN, USA), and the column was Halo ES- from Advanced Material Technology (Wilmington, DE, USA). C18 (2.1 × 100 mm, 2.7 μm,) was used. The UPLC analysis conditions were a flow rate of 0.8 mL/min, an injection amount of 2 μL, a detection wavelength of 220 nm, and a column temperature of 50 °C. 31%; 0-3 min, (A) 31-40%; 3-5 min, (A) 40-45%; It was set to 5-10 minutes. A table measuring the melittin content of each experimental group and control group is shown in Table 7.

processing conditions Melittin content (% (w/w)) Untreated purified bee venom sample 64.1 Purified bee venom sample sterilized at 121℃ for 5 minutes 51.8 Purified bee venom sample sterilized at 121℃ for 10 minutes 51.0 Purified bee venom sample sterilized at 121℃ for 15 minutes 50.7 Purified bee venom sample sterilized at 121℃ for 20 minutes 46.0

As shown in Table 7, when the purified bee venom sample was sterilized by the high-pressure sterilization process, it was confirmed that the content of melittin, the main active ingredient of bee venom, was greatly reduced by 12% or more even after only 5 minutes of the high-pressure sterilization process.

3.2 Measurement of changes in melittin content in bee venom by alcohol sterilization process

Purified bee venom samples were put into 100% distilled water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and 100% ethanol to make 1 mg/mL, vortexed for 1 minute, and filtered through a 0.2 μm membrane filter as the control group. and experimental groups, and melittin content analysis was performed in the same manner as in Example 3.1. The melittin content of each control group and experimental group is shown in Table 8.

processing conditions Melittin content (% (w/w)) 100% distilled water treated purified bee venom sample 60.7 Purified bee venom sample treated with 20% ethanol 63.7 Purified bee venom sample treated with 40% ethanol 61.8 60% ethanol-treated purified bee venom sample 57.4 Purified bee venom sample treated with 80% ethanol 3.1 100% ethanol-treated purified bee venom sample 0

As shown in Table 8, when the 20% to 40% alcohol sterilization process was performed, the melittin content was slightly increased, but this means that other components in the bee venom were eluted and the melittin was relatively increased, The results show that the alcohol sterilization process changes the components of bee venom. In addition, it was confirmed that the content of melittin, a major active ingredient of bee venom, was reduced when sterilization was performed with an alcohol sterilization process of 60% or more.

3.3 Measurement of change in melittin content in bee venom by E-beam sterilization process

Purified bee venom samples subjected to the 25 kGy E-beam sterilization process and untreated purified bee venom samples were prepared, and the melittin content of each was measured and compared in the same manner as in Example 3.1. The measured melittin content of each sample is shown in Table 9.

processing conditions Melittin content (% (w/w)) Untreated purified bee venom sample 62.1 Purified bee venom sample undergoing 25 kGy E-beam sterilization process 62.2

As shown in Table 9, in the case of sterilization by the 25 kGy E-beam sterilization process, it was confirmed that the content of melittin, the main active ingredient of bee venom, was constant before and after sterilization. Therefore, it was confirmed that the sterilization process using the E-beam did not cause a change in the active ingredient content of bee venom compared to the alcohol sterilization process or the high-pressure sterilization process.

Hereinafter, formulation examples of the composition containing bee venom of the present invention will be described, but the present invention is not intended to be limited, but merely described in detail.

Formulation Example 1. Preparation of powder

100 mg of bee venom extract of the present invention

lactose 1 g

A powder was prepared by mixing the above components and filling in airtight bags.

Formulation Example 2. Preparation of tablets

100 mg of bee venom extract of the present invention

Corn Starch 100 mg

100mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsules

100 mg of bee venom extract of the present invention

Corn Starch 100 mg

100mg milk sugar

Magnesium stearate 2 mg

After mixing the above ingredients, capsules were prepared by filling gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4. Preparation of Pills

100 mg of bee venom extract of the present invention

lactose 1.5 g

1 g of glycerin

0.5 g xylitol

After mixing the above components, it was prepared to be 4 g per pill according to a conventional method.

Formulation Example 5. Preparation of granules

100 mg of bee venom extract of the present invention

Soybean Extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, which were then filled into bags.

Claims (7)

A method of sterilizing hepatitis A virus without causing a change in melittin in bee venom, comprising irradiating bee venom with an E-beam of 25 kGy.
The method of claim 1, wherein the bee venom is live bee venom, dried bee venom, bee venom extract, or purified bee venom.
delete (a) irradiating bee venom with an E-beam of 25 kGy; and
(b) detecting hepatitis A virus in bee venom; a method for preparing bee venom sterilized with hepatitis A virus in which melittin is not induced.
The method of claim 4, wherein the step of spiking hepatitis A virus into the bee venom prior to step (a); How to manufacture. delete delete

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